Method for solid-phase synthesis of dna encoded compound library

ABSTRACT

The present invention provides a method of solid-phase synthesis of DNA-encoded compound library. The method includes following steps: a) reacting solid carrier G-1 with linker molecule L-1 to prepare L-G-1; b) reacting DNA with linker molecule L-0 to prepare L-2; c) reacting L-G-1 with L-2 to prepare L-G-2; d) removing protection group of the L-G-2 and obtaining L-G-2-1; e) reacting the L-G-2-1 with synthetic building block and performing DNA encoding; and f) removing the solid carrier and obtaining the DNA-encoded compound library. Compared with the prior art, the present invention can complete post-treatment purification of the reaction only by filtration and irrigation processes for several times. The present invention is simple to operate, can shorten the production cycle of DNA encoded compound library with more than 50%, significantly increases the production efficiency and the unicity as well as the purity of the final products.

TECHNICAL FIELD

The present invention relates to a method for solid-phase synthesis ofDNA encoded compound library.

BACKGROUND

High throughput screening (HTS) aiming at biological targets is one ofthe major means to rapidly obtain lead compounds in research anddevelopment of pharmaceuticals. However, this method imposes arestriction on efficiency and possibility of discovering lead compounds,given to a long time needed for traditional HTS of a single molecule,enoumous investment of equipments, a limited amount of library compounds(counted in millions) as well as over-long establishment of compoundlibrary in terms of several decades. DNA encoded compound library foundin recent years combine combinatorial chemistry and molecular biologyand is able to synthesis a compound library with up to hundreds ofmillion molecules within a very short time by adding a DNA tag to everycompound at a molecular level. Also, these compounds can be identifiedby gene sequencing. It largely increase size of a compound library aswell as synthesis efficiency and becomes a trend of the next generationof compound library screening technology. A extensive application inpharmaceutical industry has already started in developed countries,producing a great number of positive results (Accounts of ChemicalResearch, 2014, 47, 1247-1255).

Since every compound molecule in the DNA encoded compound library has aspecial given DNA sequence, the screening of a compound library is ableto take the DNA encoded compound library which contains ten million oreven hundred million of compounds as a whole, dissolving them into tensof micro liters to perform the high throughput screening of biologicaltargets under normal laboratory conditions. No screening equipments arenecessary and complex management system for compounds library is avoidedcompared to the traditional high throughput screening, which enormouslysaves equipment investment as well as screening cost.

In 1993, inspired by genetic DNA tag, Brenner et al utilized theencoding of beads, making every bead attached to a single compound andencoding the synthesis process into the beads, and identified compoundswhich attached to the bead by reading the code on the bead. Thissolid-phase “method for synthesis of DNA encoded combinatorialchemistry” was presented in the American chemistry society (J. Am. Chem.Soc. 1993, 115, 9812-9813). However, the method Brenner presented canonly be applied on the synthesis of polypeptide library, the need forsynthesis of small molecule compound library in the modern chemistry andpharmaceutical industries is still not meet.

Later on, Pehr B. Harbury et al., has developed another method forsolid-phase synthesis of DNA encoded compound library and utilized it onthe synthesis of polypeptide (PloS Biology year 2004, 2, page1031-1038). But the solid applied is the anion exchange resin DEAEsepharose. The forces between solid carrier and DNA mainly come fromion-ion interaction. There is thus hardly stable existence inhigh-salute system or in the system where other influencing ionsinteract, which impose a large restriction on the application ofsynthesis of small compound library.

An invention application numbered CN201210555548.3, namely “Synthesisand Screening Method and Kit for Lead Compound”, disclose a technologyin synthesis and encoding of a DNA encoded compound library. Itcomprises steps as follows. Firstly the beginning sequence of a singlestrand in DNA is connected with the synthetic building block, and theother end of the strand is series connected with special marker of thesynthesis building block in order to derive a begin synthetic buildingblock whose end marked with a single strand DNA; then based on the beginsynthetic building block, compounds are synthesized by using linearcombination reactions. In the process of synthesis, each time a newsynthesis building block is added, a special maker sequence of the newblock is series connected with the free-end of single strand DNA whichconnected to the begin synthesis building block, increasing length ofthe described the single strand DNA; finally the free-end of singlestrand DNA is connected to the end sequence and a single strand DNAencoded compound library is derived. This invention, combined withscreening and sequencing processes, is able to synthesize target leadcompound rapidly and efficiently.

The existing technology in synthesis and encoding of DNA encodedcompound library has some drawbacks and limitations, although it can bewell applied on the screening of lead compounds. Above all, the encodedDNA, to a large degree, is only available in an aqueous (or an aqueousphase containing an organic solvent which is dissolved in water)reaction. Solubility of reacting molecules in pure organic solvents ispoor so that this method can not be applied to the reaction withwater-sensitive pure organic solvents, thus limiting type of synthesisapplying DNA encoded compound library and decreasing the synthesis rateof DNA encoded compound library. Secondly, an over-dose of smallmolecule reactant is often used in order to encourage each reaction stepto react entirely but it is hard to separate the reactants completelyfrom water-based DNA encoded compound library. Finally, the separationand purification processes always contains complex procedures and alwaystake a long cycle to make sure that purification requirement of DNAcompound library is meet after the DNA library synthesis during which isbased on liquid reaction.

Nowadays, the present solid-based method for DNA encoded compoundsynthesis which is only applied on synthesis of polypeptides covers alimited type of chemical reactions. Moreover, the forces between solidcarrier and DNA mainly come from ion-ion interaction. There is thushardly stable existence in high-salute system or in the system whereother influencing ions interact, which impose a large restriction on theapplication of synthesis of small compound library.

In all, a new method for solid-phase synthesis of DNA encoded compoundlibrary is needed, with easy procedures, a low cost, a short cycle andan organic solvent suitable system.

SUMMARY

The present invention disclose a method for solid-phase synthesis of DNAencoded compound library, in order to solve the above mentionedproblems. It optimizes the method for synthesis and purificationprocesses of DNA encoded compound library, increases the structuraldiversity of small chemical molecules in DNA encoded compound library,and enlarges the scope of its application on screening of newpharmaceuticals.

Terminologies appearing in the following description are explained asfollows.

Synthetic Building Block, namely synthon, refers to a kind of smallmolecules used in the research and development of new pharmaceuticals(Western medicine and traditional Chinese medicine) which has a varietyof physical properties as well as specific chemical properties.

Solid carrier, refers to a solid material for carrying other moleculesused in a solid-phase reaction.

Linker molecule, also namely as binding molecule, refers to a kind ofcompound having a special group to connect DNA, small molecule compoundlibrary and solid carrier together.

CPG (Controlled pore Glass) refers to micro-pore glass balls made fromsilicon dioxide. It has a large number of random pores inside the ballswith the pores forming a large network. The size of the pore is namedpore diameter, the pore diameter is very stable.

The present invention provides a method for solid-phase synthesis of aDNA-encoded compound library, the method comprises the following steps:

a. reacting a solid carrier G-1 with a linker molecule L-1, separating,purifying and obtaining L-G-1;

b. reacting DNA with a linker molecule L-0, separating, purifying andobtaining L-2;

c. reacting the L-G-1 with the L-2, separating, purifying and obtainingL-G-2;

d. removing a protection group of the L-G-2 and obtaining L-G-2-1; and;

e. adopting method 1, method 2, method 3, method 4 or any combinationthereof: method 1, comprising:

reacting the L-G-2-1 with R¹, separating, purifying and obtainingL-G-2-1-1; performing DNA encoding on the L-G-2-1-1 and obtainingL-G-3-I;

or method 2, comprising:

i. reacting the L-G-2-1 with skeleton molecules, separating, purifyingand obtaining L-G-2-2; and

ii. reacting the L-G-2-2 with R¹ or reacting the R¹ with the L-G-2-2having protection group removed, separating, purifying and obtainingL-G-2-2-1; performing DNA encoding on the L-G-2-2-1 and obtainingL-G-3-1;

or method 3, comprising:

i. reacting the L-G-2-1 with skeleton molecules, separating, purifyingand obtaining L-G-2-2;

ii. reacting the R¹ with the L-G-2-2 or reacting the R¹ with the L-G-2-2having protection group removed, separating, purifying and obtainingL-G-2-2-1; performing DNA encoding on the L-G-2-2-1 and obtainingL-G-3-1; and

iii.

reacting a R² with the L-G-3-1 or reacting the R² with the L-G-3-1having protection group removed, separating, purifying and obtainingL-G-3-1-1; performing DNA encoding on the L-G-3-1-1 and obtainingL-G-3-2;

or method 4, comprising:

i. reacting L-G-2-1 with skeleton molecules, separating, purifying andobtaining L-G-2-2; performing DNA encoding on the L-G-2-2 and obtainingL-G-3-1;

ii. reacting the R2 with the L-G-3-1 or reacting the R2 with the L-G-3-1having protection group removed, separating, purifying and obtainingL-G-3-1-1; performing DNA encoding on the L-G-3-1-1 and obtainingL-G-3-2;

wherein, R¹ and R² are synthetic building blocks;

f. removing the solid carrier from L-G-3-I, L-G-3-1 and/or L-G-3-2 andobtaining DNA-encoded compound library L-D-T.

Further, in step a, the described solid carrier is selected from any oneor more of a PEG resin, a PEGA resin, an inorganic carrier and a PE thinplate.

Further, in step a, the described solid carrier is from a solid carriercontaining amino active functional group, preferably the solid carrieris

Further, in step a, the described linker molecule L-1 is selected from acompound containing any one or more of functional groups consisting ofester group, sulfur-ester group, ortho-nitrobenzyl group, coumaringroup, aromatic ketone groups, nitrine, hydroxyl group, sulfhydrylgroup, thioether group, carboxyl group, aldehyde group, amino groups,amide group, alkenyl group and alkynyl group; and the described linkermolecule L-1 is

Further, in step a, a weight ratio of the described solid carrier G-1and the described linker molecule L-1 is 1:(0.08˜0.2); preferably, aweight ratio of the described solid carrier G-1 and the described linkermolecule L-1 is 1:0.138.

Further, in step a, the reaction solvent is a halogenated hydrocarbonsolvent; preferably, reaction solvent is dichloromethane.

Further, in step a, the reaction temperature is 15˜25° C. and reactiontime is 12˜16 hours.

Further, in step a, the method of separation and purification isremoving a solvent to obtain a solid and washing the solid by DMF anddichloromethane.

Further, in step a, the described L-G-1 is

Further, in step b, the described DNA is single-strand DNA.

Further, in step b, the described linker molecule L-0 is selected from acompound containing any one or more of functional groups consisting ofester group, sulfur-ester group, ortho-nitrobenzyl group, coumaringroup, aromatic ketone groups, nitrine, hydroxyl group, sulfhydrylgroup, thioether group, carboxyl group, aldehyde group, amino groups,amide group, alkenyl group and alkynyl group; preferably, the describedlinker molecule is

Further, in step b, reaction is carried out with a condensing agent.

Further, in step b, the condensing agent is2-Chloro-4,6-dimethoxy-1,3,5-triazine.

Further, in step b, the molar ratio of DNA and the linker molecule L-0is 1:50˜300 and the molar ratio of the linker molecule L-0 and thecondensing agent is 1:1˜2; preferably, the molar ratio of DNA and thelinker molecule L-0 is 1:100 and the molar ratio of the linker moleculeL-0 and the condensing agent is 1:1.

Further, in step b, the reaction temperature is 15˜25° C. and reactiontime is 12˜16 hours.

Further, in step b, the method of separation and purification comprises:adjusting pH to 4˜5, adding ethanol, precipitating at −20° C., washingand obtaining a solid.

Further, in step b, the L-2 is:

Further, in step c, the reaction is carried out with an organic solvent;and a reaction solvent is pyridine.

Further, in step c, a molar ratio of L-G-1 and L-2 is 2˜10:1 and theweigh-to-volume ratio of L-G-1 and the pyridine is 1:1 mg/μL.

Further, in step c, the reaction temperature is 20˜45° C., and reactiontime is 14˜30 hours; preferably, the reaction temperature is 40° C. andthe reaction time is 21 hours.

Further, in step c, the method of separation and purification is:removing the solvent to derive solids and washing solids with water andTEAA buffer solution.

Further, in step c, the described L-G-2 is

Further, in step d, the protection group is amino protection group,preferably, the protection group is Fluorenylmethoxycarbonyl group(FMOC).

Further, in step d, the protection group is removed of L-G-2 withpiperidine and nitrogen-containing solvent, wherein a molar volume ratioof the described L-G-2 and piperidine is 1:(100˜10000) mol/L and avolume ratio of the piperidine and the nitrogen-containing solvent is(1˜4):(5˜20).

Further, in step d, the described L-G-2-1 is

Further, in step e, the method of DNA encoding comprises: connectingmarker sequence i with DNA of L-G-2-1-1, L-G-2-2-1, L-G-3-1-1 or L-G-2-2in series and marking R¹ specifically with the marker sequence i;wherein R^(i) is a synthetic building block, i=1 or 2; connecting themarker sequence i on DNA or marker sequence (i−1) in series when a newR^(i) is added each time during the synthesis; and connecting a terminalsequence on the marker sequence i after the synthesis is completed.

Further, in step e, the described R¹ and R² are respectively orsimultaneously selected from polyfunctional group compounds, and thepolyfunctional groups are independently selected from any two or more ofgroups consisting of amino, carboxyl, aldehyde group, alkenyl, alkynyl,halogen, azide group, hydroxyl group, sulfhydryl group and phenyl group;preferably, the described R¹ and R² are respectively or simultaneouslyselected from amino acid, substituted or unsubstituted carboxylic acids,substituted or unsubstituted amines, substituted or unsubstitutedalkenes, substituted or unsubstituted alkynes, substituted orunsubstituted aldehydes, isocyanate; and the described R¹ and R² arerespectively or simultaneously selected from isocyanate, benzyl alcoholor benzoic acid.

Further, in step e, the described skeleton molecules contain any one ormore of groups consisting of hydroxyl group, amino group, carboxylgroup, cyanate group and aldehyde group.

Further, in step e, the described skeleton molecules are selected fromany one or more of molecules consisting of 3-(4-hydroxyphenyl)propionicacid, 4-aminobenzoic acid, dl-4-hydroxyphenylglycine, FMOC-glycine,FMOC-1-phenylalanine, t-butysocynide, cyclohexyl isocyanide, 3-methylbutyraldehyde, cyclopentyl aldehyde and

Further, the reaction is carried out under the condition of an organicamine and an organic solvent; preferably, the reaction is carried outunder the condition of triethylamine and dimethylformamide.

Further, in method 1 of step e, a molar ratio of L-G-2-1, R₁ and theorganic amine is 1:(50˜500):(50˜500), a molar volume ratio of L-G-2-1and the organic solvent is 1:(50˜500) nmol/μL; and the molar ratio ofL-G-2-1, R₁ and the organic amine is 1:150:150 and the molar volumeratio of L-G-2-1 and the organic solvent is 1:240 nmol/pt.

Further, in method 1 of step e, the reaction temperature is 25° C.˜30°C. and the reaction time is 12˜16 hours.

Further, in method 1 of step e, the method of separation andpurification is: removing the solvent to derive solids, washing solidswith water and TEAA buffer solution.

Further, in method 2 or method 3 of step e, the reaction in the step iis carried out under the condition of a condensing agent, an organicamine and an organic solvent; in the method 4 of the step e, thereaction in the step i is carried out under the condition of organicsolvent; and the reaction is carried out under the condition of2-(7-azobenzene and -triazole)-N,N,N′,N′-tetramethylureahexafluorophosphate, N,N-ethyldiisopropylamine and the nitrogencontaining solvent;

a molar ratio of the described L-G-2-1, the skeleton molecule, thecondensing agent and the organic amine is 1:50˜500:50˜500:50˜500; amolar volume ratio of the described compounds L-G-2-1 and the nitrogensolvent is 50˜500 nmol/μL.

Further, in method 2, method 3 or method 4 of step e, the reactiontemperature in step i is 25° C.˜30° C. and reaction time is 12˜16 hours.

Further, in method 2, method 3 or method 4 of step e, the method ofseparation and purification in step i is: removing the solvent to derivesolids, washing solids with water and TEAA buffer solution.

Further, in method 2, method 3 or method 4 of step e, the reaction inthe step ii is carried out under the condition of an organic amine ororganic phosphine and an organic solvent; and the described organicamine is selected from N,N-ethyldiisopropylamine or triethylamine, thedescribed organic phosphine is triphenylphosphine and the describedorganic solvent is selected from halohydrocarbon solvents, etherssolvents and nitrogen solvents; and the described ethers solvent istetrahydrofuran and the nitrogen solvent is N,N-dimethylformamide.

Further, in method 2, method 3 or method 4 of step e, the reactiontemperature in step ii is 25° C.˜30° C. and reaction time is 12˜16hours.

Further, in method 2, method 3 or method 4 of step e, the method ofseparation and purification in the step ii is: removing the solvent toobtain a solid, washing the solid with water and TEAA buffer solution.

Further, in method 2, method 3 or method 4 of step e, the method ofremoving the protection group from L-G-2-2 or L-G-3-1 is: addingpiperidine into L-G-2-2 or L-G-3-1, stirring at 25° C.˜30° C. for 2˜6hours, removing the solvent to obtain a solid, and washing the solidwith water and TEAA buffer solution.

Further, in method 3 or method 4 of step e, the reaction with R² iscarried out under the condition of organic amines and organic solvents;and the described organic amine is selected fromN,N-ethyldiisopropylamine or triethylamine, and the described organicsolvent is selected from halohydrocarbon solvents.

Further, in method 3 or method 4 of step e, the temperature of reactionwith R² is 25° C.˜30° C. and the time of reaction with R² is 12˜16hours.

Further, in method 2, method 3 or method 4 of step e, the method ofseparation and purification in step iii is: removing the solvent toobtain a solid, and washing the solid with water and TEAA buffersolution.

Further, in step e, the described L-G-3-I is

-   -   the described L-G-2-2 is

the described L-G-3-1 is:

wherein, R^(j) is a group able to form isocyanate.

Further, in step f, the method of removing the solid carrier comprises:

i. taking L-G-3-I, L-G-3-1 and/or L-G-3-2, adding an alkaline forreaction, separating and purifying; or

ii. taking L-G-3-I, L-G-3-1 and/or L-G-3-2, adding PBS buffer solution,performing decomposition reaction under light source, separating andpurifying.

Further, in step i. the described alkaline is an inorganic alkaline;reaction temperature is 30° C.˜60° C.; reaction time is 1˜10 hours;preferably, the described alkaline is aqueous ammonia; reactiontemperature is 55° C.; reaction time is 1 hour.

Further, in step ii, the wavelength of the light is 365 nm; temperatureof the decomposition reaction is 25° C.˜40° C.; the reaction time is 1˜8hours.

Further, in step i and/or step ii,

the method of separation and purification is: washing with water andTEAA buffer solution to obtain a filtrate; adding ethanol afterconcentrating the filtrate, precipitating at −20° C., centrifugating,obtaining a solid and washing the solid.

Further, in step f, the described L-D-T is

wherein, R^(j) is a group able to form isocyanate; R_(1-a) is —CH₂— or—C₆H₅CH₂CH—; R_(1-b) is n-butyl, isobutyl, tertiary butyl or cyclohexyl;R_(1-a) is n-propyl, isopropyl or cyclopentyl; R^(k) is a group producedby amino group reacting with any one of carboxylic acid, aldehyde andisocyanate.

The present invention provides a kind of DNA encoded compound library,and it has general formula like formula I:

wherein,

is a skeleton molecule in the compound library;

R^(i′) is selected from hydrogen or synthetic building blocks;

E is selected from amino group, alkenyl, alkynyl, amide, ester,thioether group or azide;

A is selected from a hydroxyl group or a sulfhydryl group;

B is selected from

wherein R is hydrogen, C1˜C8 alkyl group, C1˜C8 alkenyl group or a groupforming a cycle with atoms in B₁; B₁ is selected from substituted orunsubstituted alkynyl, amino, carboxyl, aldehyde, azide or sulfhydryl;and

is DNA.

Further, the described skeleton molecule contains any one or more ofgroups consisting of hydroxyl, amino, carboxyl, cyano group and aldehydegroup.

Further, the described skeleton molecule is any one or more of followingmolecules consisting of 3-(4-hydroxyphenyl)propionic acid,4-aminobenzoic acid, dl-4-hydroxyphenylglycine, FMOC-glycine,FMOC-1-phenylalanine, t-butylisocynide, cyclohexyl isocyanide, 3-methylbutyraldehyde, cyclopentyl aldehyde and

Further, molecule A has 10˜50 atoms.

Further, the library contains the following DNA encoded compoundlibrary:

wherein, R^(j) is a group able to form isocyanate; R_(1-a) is —CH₂— or—C₆H₅CH₂CH—; R_(1-b) is n-butyl, isobutyl, tertiary butyl or cyclohexyl;R_(1-a) is n-propyl, isopropyl or cyclopentyl; R^(k) is a group producedby amino group reacting with any one of carboxylic acid, aldehyde andisocyanate.

Compared with the existed method for liquid phase synthesis of DNAencoded compound library, the present invention can completepost-treatment purification of the reaction only by filtration andirrigation processes for several times. The present invention is simpleto operate, can shorten the production cycle of DNA encoded compoundlibrary with more than 50%, significantly increases the productionefficiency and the unicity as well as the purity of the final products.Besides, the present invention has a high economic value, which issuitable for industrial application.

Specifically, technical solutions are further shown as follows.

1. Adding 3 mL of dichloromethane solution containing compound L-1 (69mg, 0.1 mmol, from Aldrich) to G-1 (0.5 g, loading: 32 μmol/g) andallowing reaction overnight under room temperature; removing the solventby filtration after the reaction to obtain a solid; washing the solidrespectively with DMF (2 mL×3) and dichloromethane (2 mL×3) until nocompound L-1 can be found in the solution with LC-MS; L-G-1 is obtained.

Taking a portion of L-G-1, removing Fmoc protection group by piperidine,determining that the loading of CPG is 14.5 μmol/g by the UV absorptionof free amino groups.

2. Dissolving single strand DNA into 30 μL NaHCO₃ buffer solution,adding successively 20 mL of DMSO solution of L-0 (1.0 mg, 3.0 μmol,manufacturer: Aldrich) and 10 μL water solution of DMT-MM solution (0.83mg, 3.0 μmol); allowing reaction overnight under room temperature;adjusting pH to 4˜5 with 3 mol/L HCl solution after the above mentionedreaction is finished, adding 240 μL ethanol and precipitating at −20° C.for 2 hours, centrifugating to obtain a solid; washing the solid with85% ethanol to obtain L-2.

3. Adding 90 μL aqueous solution of L-2 (28 nmol) and 10 μL pyridineinto L-G-1 (10 mg, 140 nmol), and allowing reaction with stirring for 21hours at temperate of 40° C.; filtrating the solution after the abovementioned reaction, and removing the solvent and obtain a solid; washingthe solid respectively with distilled water and 0.1 mol/L TEAA buffersolution for 3 times to obtain L-G-2.

4. Adding nitrogen solvent and pyridine into L-G-2, allowing reactionwith stirring for 4˜6 hours at 25° C.˜30° C.; filtrating the solution toremove the solvent and to get the filtrated cake; washing the filtratedcake respectively with distilled water and 0.1 mol/L TEAA buffersolution for 3 times to obtain L-G-2-1.

The molar volume ration of the described L-G-2 and pyridine is1:(1˜4)mol/mL; the volume ratio of pyridine and nitrogen-containedsolvent is (1˜4):(5˜20).

5. Adding molecule skeleton containing carboxylic acid functional group,O-(7-azabenzotriazole-1-yl)-N, N,N′,N′-trtramethyluroniumhexafluorophosphate, N,N-ethyldiisopropylamine as well ashalohydrocarbon derivative solvent into L-G-2-1 and allowing reactionwith stirring for 12˜16 hours at 25° C.˜30° C.; filtrating the solutionto remove the solvent to obtain a filtrated cake; washing the filtratedcake respectively with distilled water and 0.1 mol/L TEAA buffersolution for 3 times to obtain L-G-2-2.a.

The molar ration of the L-G-2-1, molecule skeleton containing carboxylicacid functional group, O-(7-azabenzotriazole-1-yl)-N,N,N′,N′-trtramethyluronium hexafluorophosphate andN,N-ethyldiisopropylamine is 1:(1˜2):(1˜2):(2˜4); the weight volumeratio of the described L-G-2-1 and halohydrocarbon derivative solvent is1:(9˜20)g/mL;

6. Adding R^(i) (R^(i) is synthetic building block, selected fromisocyanate, benzyl alcohol or benzoic acid, i=1, 2 or 3),N,N-ethyldiisopropylamine and halohydrocarbon derived solvents intoL-G-2-2.a; allowing reaction with stirring for 12˜16 hours at 25° C.˜30°C.; filtrating the solution to remove the solvent and obtaining afiltrated cake; washing the filtrated cake respectively with distilledwater and 0.1 mol/L TEAA buffer solution for 3 times to obtainL-G-2-2-1.a;

the DNA encoded technology, disclosed by file application number:CN201210555548.3, named “Synthesis and Screening Method and Kit for ALead Compound”, is adopted after the purification process of each stepfor reactions of Synthetic Building Blocks, in order to performbio-enzyme catalyzed DNA encoding on R¹, R² and R³ in present inventedcompound library and to obtain L-G-3-1.a.

7. Removing CPG from the L-G-3-1.a by using alkaline removal method orlight removal method to obtain DNA encoded compound library L-D-T-1.

Obviously, based on the above mentioned invention, different kinds ofother modifications, substitutions or changes can be made according tonormal technological knowledge and common tool in the present field butare still within the technological idea of the present invention.

To give a further detailed description of the present invention, someembodiments are given below. However, these embodiments should not beunderstood as the limits of scope that the present invention covers. Anytechnological solution based on the described content above lies in thescope of the present invention.

DETAILED DESCRIPTION

The raw materials and equipments used in the present invention are knownproducts and can be purchased in the market.

Some abbreviations are given:

Fmoc: fluorenylmethoxycarbonyl group;

DMF: N,N-dimethylformamide;

DMSO: dimethyl sulfoxide;

DMT-MM: 2-chloro-4,6-dimethoxy-1,3,5-triazine;

TEAA: triethylammonium acetate; and

DETA: ethyldiisopropylamine.

Embodiment 1

(1). The preparation of L-G-1

adding 3 mL of dichloromethane solution of compound L-1 (69 mg, 0.1mmol, manufacturer: Aldrich) to G-1 (0.5 g, starting loading of CPG: 32μmol/g, derived from Shanghai Lingjiang Industrial Development Co., Ltd)and allowing reaction overnight under room temperature; removing thesolvent by filtration after the reaction to obtain a solid; washing thesolid respectively with DMF (2 mL×3) and dichloromethane (2 mL×3) toobtain L-G-1.

Taking L-G-1, removing Fmoc protection group with piperidine, anddetermining that the loading of L-G-1 is 14.5 μmol/g by measuring UVabsorption of the Fmoc-removed product, the productivity is 91%.

(2) The preparation of L-2

dissolving single strand DNA (32.0 nmol, molar weight is 7663.9)(sequence of the single strand DNA is GGAGCTTGTGAATTCTGGCACTCG) into 30μL NaHCO₃ buffer solution, adding successively 20 mL DMSO solution ofL-0 (1.0 mg, 3.0 μmol, manufacturer: Aldrich) and 10 μL water solutionof DMT-MM solution (0.83 mg, 3.0 μmol); allowing reaction overnightunder room temperature; adjust pH to 4˜5 with 3 mol/L HCl solution afterthe above mentioned reaction is finished; adding 240 μL ethanol andprecipitating at −20° C. for 2 hours, and centrifugating to obtain asolid; washing the solid with 85% ethanol to obtain L-2, and determiningthat the amount of substance of L-2 is 28.0 nmol using OD ultravioletabsorption, the productivity is 80%.

MS(ESI) m/z 7979.6 (M+1)⁺.

(3) The preparation of L-G-2

adding 90 μL aqueous solution of L-2 (28 nmol) and 10 μL pyridine intoL-G-1 (10 mg, 140 nmol), and allowing reaction with stirring for 21hours at 40° C.; filtrating the solution after the above mentionedreaction, removing the solvent to obtain a solid; washing the solidrespectively with distilled water and 0.1 mol/L TEAA buffer solution for3 times to obtain L-G-2.

(4) The preparation of L-G-2-1

Adding DMF (160 μL) and pyridine (40 μL) into L-G-2 (8.0 mg), andallowing react with stirring for 6 hours at 25° C.˜30° C.; filtratingthe solution and removing the solvent to obtain a filtrated cake;washing the filtrated cake respectively with distilled water and 0.1mol/L TEAA buffer solution for 3 times to obtain L-G-2-1.

Taking part of L-G-2-1 (3.0 mg), adding 150 μL of strong aqueousammonia, and heating the solution to 55° C. and reacting for 1 hour toremove the solid carrier, removing the solvent by reducing the pressureafter filtration process, washing the solid respectively with distilledwater and 0.1 mol/L TEAA buffer solution for 3 times, adding 250 μLethanol and 100 μL acetic acid-sodium acetate buffer solution (pH=4.7,0.5 mol/L) to the solid and precipitating at −20° C. to obtain the DNAin L-G-2-1, determining that the amount of substance of DNA in L-G-2-1is 6.0 nmol by using OD ultraviolet absorption, the productivity is 65%.

MS(ESI) m/z 8251.0 (M+1)⁺.

(5) the preparation of L-G-2-2.a

Adding molecule skeleton containing carboxylic acid,O-(7-azabenzotriazole-1-yl)-N, N,N′,N′-trtramethyluroniumhexafluorophosphate, DIEA as well as DMF into L-G-2-1 and allowing reactwith stirring for 16 hours at 25° C.˜30° C.; filtrating the solution andremoving the solvent to obtain a filtrated cake; washing the filtratedcake respectively with distilled water and 0.1 mol/L TEAA buffersolution for 3 times to obtain L-G-2-2.a.

More specifically,

Adding 4-Aminobenzoic Acid (4.15 mg, Manufacturer: Alfa),O-(7-Azabenzotriazole-1-yl)-N, N,N′,N′-trtramethyluroniumhexafluorophosphate (6.9 mg manufacturer: Alfa), DIEA (20 μL) as well asDMF (60 μL) into L-G-2-1 (20 mg) and allowing reaction with stirring for12˜16 hours at 25° C.˜30° C.; filtrating the solution and removing thesolvent to obtain a filtrated cake; washing the filtrated cakerespectively with distilled water and 0.1 mol/L TEAA buffer solution for3 times to obtain L-G-2-2.a.

Taking part of L-G-2-2.a (3.0 mg), adding 150 μL of strong aqueousammonia and heating the solution to 55° C. for 1 hour to remove thesolid carrier, removing the solvent by reducing the pressure afterfiltration process, washing respectively with distilled water and 0.1mol/L TEAA buffer solution for 3 times. Adding 250 μL ethanol and 100 μLacetic acid-sodium acetate buffer solution (pH=4.7, 0.5 mol/L) to thesolid and precipitating at −20° C. to obtain the DNA in L-G-2-2,determining that the amount of substance of DNA in L-G-2-1 is 4.5 nmolby using OD ultraviolet absorption, the productivity is 75%.

MS(ESI) m/z 8369.7 (M+1)+.

(6) The preparation of L-G-3-1.a

adding R^(j′) (R^(j′) is synthetic building block and selected fromsubstituted isocyanate), DIEA and DMF into L-G-2-2.a; allowing reactionwith stirring for 16 hours at 25° C.˜30° C.; filtrating the solution andremoving the solvent to obtain a filtrated cake; washing the filtratedcake respectively with distilled water and 0.1 mol/L TEAA buffersolution for 3 times to obtain L-G-2-2-1.a;

referencing to the DNA encoded method disclosed in the patent named“Synthesis and Screening Method and Kit for Lead Compound” (applicationnumber: CN201210555548.3), L-G-2-1-1.a is performed DNA encoding insolid-phase condition (single strand DNA sequence isGGAGCTTGTGAAATCTGGCACTCG) to obtain L-G-3-1.a;

after the encoding, washing the solid respectively with 0.1 mol/L TEAAbuffer solution (4×100 μL) and distilled water (4×100 μL), finallywashing with 100 μL distilled water; taking a part of the solid washing,adding 50 μL of strong aqueous ammonia, and allowing reaction for 1 hourat 55° C.; washing the solid with 0.1 mol/L TEAA buffer solution anddistilled water for 3 times; precipitating the filtrated solution byethanol, and freeze-drying for agarose electrophoresis test.

(7) The synthesis of compound library L-D-T-1

removing CPG from L-G-3-1.a by alkaline removal method or light removalmethod to obtain the DNA encoded compound library L-D-T-1.

Embodiment 2

(1) L-G-1 is obtained according to the preparation method for L-G-1 inembodiment 1;

(2) L-2 is obtained according to the preparation method for L-2 inembodiment 1;

(3) L-G-2 is obtained according to the preparation method for L-G-2 inembodiment 1;

(4) L-G-2-1 is obtained according to the preparation method for L-G-2-1in embodiment 1; and

(5) the preparation of L-G-3-1.b

adding 2-ethylphenyl isocyanate (1.47 mg, manufacturer: Alfa),triethylamine (5 μL) as well as DMF (15 μL) into L-G-2-1 (5 mg) andreacting for 16 hours at 25° C.˜30° C.; filtrating the solution andremoving the solvent to obtain a filtrated cake; washing the filtratedcake respectively with distilled water and 0.1 mol/L TEAA buffersolution for 3 times to obtain L-G-2-1-1.b;

taking part of L-G-2-1-1.b (2.0 mg), adding 150 μL of strong aqueousammonia, and heating the solution to 55° C. for 1 hour to remove thesolid carrier. Removing the solvent by reducing the pressure afterfiltration process, washing respectively with distilled water and 0.1mol/L TEAA buffer solution for 3 times. Adding 250 μL ethanol and 100 μLacetic acid-sodium acetate buffer solution (pH=4.7, 0.5 mol/L) to thesolid, precipitating at −20° C. to obtain DNA in L-G-2-1-1.b. The amountof substance is 2.5 nmol by using OD ultraviolet absorptionquantitation, the productivity is 63%.

MS(ESI) m/z 8395.4 (M+1)⁺.

Referencing to the DNA encoded method disclosed in the patent named“Synthesis and Screening Method and Kit for Lead Compound” (applicationnumber: CN201210555548.3), L-G-2-1-1.b is performed DNA encoding insolid-phase condition (single strand DNA sequence isGGAGCTTGTGAAATCTGGCACTCG) to obtain L-G-3-1.b;

after the encoding, washing the solid respectively with 0.1 mol/L TEAAbuffer solution (4×100 μL) and distilled water (4×100 μL), finallywashing with 100 μL distilled water; taking a part of solids after theirrigation, adding 50 μL of strong aqueous ammonia, and reacting for 1hour at 55° C.; washing the obtained solid with 0.1 mol/L TEAA buffersolution and distilled water for 3 times; precipitating the filtratedsolution by ethanol, and freeze-drying for agarose electrophoresis test.

(6) L-D-T-2 is obtained according to the preparation method inembodiment 1.

Embodiment 3

(1) L-G-1 is obtained according to the preparation method for L-G-1 inembodiment 1;

(2) L-2 is obtained according to the preparation method for L-2 inembodiment 1;

(3) L-G-2 is obtained according to the preparation method for L-G-2 inembodiment 1;

(4) L-G-2-1 is obtained according to the preparation method for L-G-2-1in embodiment 1; and

(5) the preparation of L-G-2-2.c

adding 4-aminobenzoic acid (4.15 mg, manufacturer: Alfa),O-(7-azabenzotriazole-1-yl)-N, N,N′,N′-trtramethyluroniumhexafluorophosphate (6.9 mg, manufacturer: Alfa), DIEA (20 μL) as wellas DMF (60 μL) into L-G-2-1 (20 mg), and reacting with stirring for 16hours at 25° C.˜30° C.; filtrating the solution to remove the solventand to obtain a filtrated cake; washing the filtrated cake respectivelywith distilled water and 0.1 mol/L TEAA buffer solution for 3 times toobtain L-G-2-2. c;

taking part of L-G-2-2.c (2.0 mg), adding 150 μL of strong aqueousammonia, and heating the solution to 55° C. for 1 hour to remove thesolid carrier. Removing the solvent by reducing the pressure afterfiltration, washing the solid respectively with distilled water and 0.1mol/L TEAA buffer solution for 3 times. Adding 250 μL ethanol and 100 μLacetic acid-sodium acetate buffer solution (pH=4.7, 0.5 mol/L) to thesolid, precipitating at −20° C. to obtain DNA in L-G-2-2.c, the amountof substance of the obtained DNA in L-G-2-2.c is 3.0 nmol by using ODultraviolet absorption quantification, the productivity is 75%.

MS (ESI) m/z 8369.7 (M+1)⁺.

(6) The preparation of L-G-3-1.c

adding benzoic acid (2.0 mg), O-(7-azabenzotriazole-1-yl)-N,N,N′,N′-trtramethyluronium hexafluorophosphate (6.9 mg, manufacturer:Alfa), DIEA (20 μL) as well as DMF (60 μL) into L-G-2-1 (20 mg), andreacting with stirring for 16 hours at 25° C.˜30° C.; filtrating thesolution to remove the solvent and obtaining a filtrated cake; washingthe filtrated cake respectively with distilled water and 0.1 mol/L TEAAbuffer solution for 3 times to obtain L-G-2-2-1.c;

taking part of L-G-2-2-1.c (2.0 mg), adding 150 μL of strong aqueousammonia, and heating the solution to 55° C. for 1 hour to remove thesolid carrier. Removing the solvent by reducing the pressure afterfiltration process, washing the solid respectively with distilled waterand 0.1 mol/L TEAA buffer solution for 3 times. Adding 250 μL ethanoland 100 μL acetic acid-sodium acetate buffer solution (pH=4.7, 0.5mol/L) to the solid, precipitating at −20° C. to obtain DNA inL-G-2-2-1.c, the amount of substance of the obtained DNA in L-G-2-2-1.cis 1.5 nmol by using OD ultraviolet absorption quantification, theproductivity is 38%.

MS(ESI) m/z 8473.1 (M+1)⁺.

Referencing to the DNA encoded method disclosed in the patent named“Synthesis and Screening Method and Kit for Lead Compound” (applicationnumber: CN201210555548.3), L-G-2-2-1.c is performed DNA encoding insolid-phase condition (single strand DNA sequence isGGAGCTTGTGAAATCTGGCACTCG) to obtain L-G-3-1.c;

after encoding, washing the solid respectively with 0.1 mol/L TEAAbuffer solution (4×100 μL) and distilled water (4×100 μL), finallywashing the solid with 100 μL distilled water; taking a part of solidthe irrigation, adding 50 μL of strong aqueous ammonia, and reacting for1 hour at 55° C.; washing the obtained solid with 0.1 mol/L TEAA buffersolution and distilled water for 3 times; precipitating the filtratedsolution by ethanol, and freeze-drying for agarose electrophoresis test.

(7) L-D-T-3 is obtained according to the preparation method of L-D-T-1in embodiment 1.

Embodiment 4

(1) L-G-1 is obtained according to the preparation method for L-G-1 inembodiment 1;

(2) L-2 is obtained according to the preparation method for L-2 inembodiment 1;

(3) L-G-2 is obtained according to the preparation method for L-G-2 inembodiment 1;

(4) L-G-2-1 is obtained according to the preparation method for L-G-2-1in embodiment 1; and

(5) the preparation of L-G-2-2.d

adding S-2 (17.4 mg, manufacturer: Alfa), O-(7-azabenzotriazole-1-yl)-N,N,N′,N′-trtramethyluronium hexafluorophosphate (6.9 mg, manufacturer:Alfa), DIEA (20 μL) as well as DMF (60 μL) into L-G-2-1 (20 mg) andreacting with stirring for 16 hours at 25° C.˜30° C.; filtrating thesolution to remove the solvent and obtaining a filtrated cake; washingthe filtrated cake respectively with distilled water and 0.1 mol/L TEAAbuffer solution for 3 times to obtain L-G-2-2.d;

taking part of L-G-2-2.d (2.0 mg), adding 150 μL of strong aqueousammonia, and heating the solution to 55° C. for 1 hour to remove thesolid carrier. Removing the solvent by reducing the pressure afterfiltration process, washing the solid respectively with distilled waterand 0.1 mol/L TEAA buffer solution for 3 times. Adding 250 μL ethanoland 100 μL acetic acid-sodium acetate buffer solution (pH=4.7, 0.5mol/L) to the solid, precipitating at −20° C. to obtain DNA inL-G-2-2.d, the amount of substance of the obtained DNA in L-G-2-2.d is2.0 nmol by using OD ultraviolet absorption quantification, theproductivity is 50%.

MS(ESI) m/z 8698.0 (M+1)⁺.

(6) The preparation of L-G-3-1.d

adding piperidine (5 μL) into L-G-2-2.d (15 mg) and reacting withstirring for 6 hours at 25° C.˜30° C.; filtrating the solution to removethe solvent and obtaining a filtrated cake; washing the filtrated cakerespectively with distilled water and 0.1 mol/L TEAA buffer solution for3 times to obtain a solid; adding R^(j′) (R^(j′) is selected fromisocyanate substituted by R^(j), 1.5 mg), triethylamine (10 μL) as wellas DMF (100 μL) into the solid; reacting with stirring for 16 hours at25° C.˜30° C.; filtrating the solution to remove the solvent andobtaining a filtrated cake; washing the filtrated cake respectively withdistilled water and 0.1 mol/L TEAA buffer solution for 3 times to obtainL-G-2-2-1.d;

referencing to the DNA encoded method disclosed in the patent named“Synthesis and Screening Method and Kit for Lead Compound” (applicationnumber: CN201210555548.3), L-G-2-2-1.d is performed DNA encoding insolid-phase condition (single strand DNA sequence isGGAGCTTGTGAAATCTGGCACTCG) to obtain L-G-3-1.d;

after encoding, washing the solid respectively with 0.1 mol/L TEAAbuffer solution (4*100 μL) and distilled water (4*100 μL), finallywashing with 100 μL distilled water; taking a part of the solid afterthe irrigation, adding 50 μL of strong aqueous ammonia, and reacting for1 hour at 55° C.; washing the obtained solid with 0.1 mol/L TEAA buffersolution and distilled water for 3 times; precipitating the filtratedsolution by ethanol, and freeze-drying for agarose electrophoresis test.

(7) Synthesis of L-D-T-4 compound library

taking part of L-G-3-1.d (3.0 mg), adding 150 μL of strong aqueousammonia, and heating the solution to 55° C. for 1 hour to remove thesolid carrier. Removing the solvent by reducing the pressure afterfiltration process, washing the solid respectively with distilled waterand 0.1 mol/L TEAA buffer solution for 3 times. Adding 250 μL ethanoland 100 μL acetic acid-sodium acetate buffer solution (pH=4.7, 0.5mol/L) to the solid, and precipitating at −20° C. to obtain L-D-T-4.

Embodiment 5

(1) L-G-1 is obtained according to the preparation method for L-G-1 inembodiment 1;

(2) L-2 is obtained according to the preparation method for L-2 inembodiment 1;

(3) L-G-2 is obtained according to the preparation method for L-G-2 inembodiment 1;

(4) L-G-2-1 is obtained according to the preparation method for L-G-2-1in embodiment 1; and

(5) the preparation of L-G-2-2.e

adding S-1, S-2 and S-3 as well as DMF into L-G-2-1 and reacting withstirring for 16 hours at 25° C.˜30° C.; filtrating the solution toremove the solvent and obtaining a filtrated cake; washing the filtratedcake respectively with distilled water and 0.1 mol/L TEAA buffersolution for 3 times to obtain L-G-2-2.e.

Specifically,

adding S-1 (FMOC-glycine, manufacturer: Alfa, 14.8 mg), S-2(t-butylisocynide, manufacturer: Alfa, 4.2 mg) and S-3(isovaleraldehyde, manufacturer: Alfa, 4.3 mg) as well as DMF (100 μL)into L-G-2-1 and reacting with stirring for 16 hours at 25° C.˜30° C.;filtrating the solution to remove the solvent and obtaining a filtratedcake; washing the filtrated cake respectively with distilled water and0.1 mol/L TEAA buffer solution for 3 times to obtain L-G-2-2. e;

taking part of L-G-2-2-1.e (2.0 mg), adding 150 μL of strong aqueousammonia to, and heating the solution to 55° C. for 1 hour to remove thesolid carrier. Removing the solvent by reducing the pressure afterfiltration process, finally washing the solid respectively withdistilled water and 0.1 mol/L TEAA buffer solution for 3 times. Adding250 μL ethanol and 100 μL acetic acid-sodium acetate buffer solution(pH=4.7, 0.5 mol/L) to the solid, precipitating at −20° C. to obtain DNAin L-G-2-2.e, the amount of substance of DNA in L-G-2-2.e is 38 nmol byusing OD ultraviolet absorption quantitation, the productivity is 76%.

MS(ESI) m/z 9111.4 (M+1)⁺.

(6) The preparation of L-G-3-1.e

referencing to the DNA encoded method disclosed in the patent named“Synthesis and Screening Method and Kit for Lead Compound” (applicationnumber: CN201210555548.3), L-G-2-2-1.e is performed DNA encoding insolid-phase condition (single strand DNA sequence isGGAGCTTGTGAAATCTGGCACTCG) to obtain L-G-3-1.e;

after encoding, washing the solid respectively with 0.1 mol/L TEAAbuffer solution (4×100 μL) and distilled water (4×100 μL), finallywashing with 100 μL distilled water; taking a part of the solid afterthe irrigation, adding 50 μL of strong aqueous ammonia, and reacting for1 hour at 55° C.; washing the obtained solid with 0.1 mol/L TEAA buffersolution and distilled water for 3 times; precipitating the filtratedsolution by ethanol, freeze-drying for agarose electrophoresis test;

adding piperidine (40 μL) into L-G-2-2-1.e (10 mg) and reacting withstirring for 6 hours at 25° C.˜30° C.; filtrating the solution to removethe solvent and obtaining a filtrated cake; washing the filtrated cakerespectively with distilled water and 0.1 mol/L TEAA buffer solution for3 times to obtain a solid; adding R^(k′) (R^(k′) is selected fromsynthetic building block containing carboxylic acids, aldehydes orisocyanates), O-(7-azbenzotriazole-1-yl)-N, N,N′,N′-tetramethyluroniumhexafluorophosphate (6.1 mg, manufacturer: Alfa), DIEA (20 μL) as wellas DMF (60 μL) into the solid; reacting with stirring for 16 hours at25° C.˜30° C.; filtrating the solution to remove the solvent andobtaining a filtrated cake; washing the filtrated cake respectively withdistilled water and 0.1 mol/L TEAA buffer solution for 3 times to obtainL-G-3-1-1.e;

referencing to the DNA encoded method disclosed in the patent named“Synthesis and Screening Method and Kit for Lead Compound” (applicationnumber: CN201210555548.3), L-G-3-1-1.e is performed DNA encoding insolid-phase condition (single strand DNA sequence isGGAGCTTGTGAAATCTGGCACTCG) to obtain L-G-3-1.e; after encoding, washingthe solid respectively with 0.1 mol/L TEAA buffer solution (4×100 μL)and distilled water (4×100 μL), finally washing with 100 μL distilledwater; taking a part of the solid after the irrigation, adding 50 μL ofstrong aqueous ammonia, and reacting for 1 hour at 55° C.; washing theobtained solid with 0.1 mol/L TEAA buffer solution and distilled waterfor 3 times; precipitating the filtrated solution by ethanol, andfreeze-drying for agarose electrophoresis test; and

(7) synthesis of L-D-T-5 compound library

taking part of L-G-3-1.e (2.0 mg), adding 40 μL of aqueous ammonia, andheating the solution to 55° C. for 1 hour to remove the solid carrier.Washing the solid respectively with distilled water and 0.1 mol/L TEAAbuffer solution for several times to obtain filtrated solution;concentrating the filtrated solution, and adding appropriate ethanol,precipitating at −20° C. and centrifugating to obtain a solid; washingthe solid with 85% ethanol to obtain DNA encode compound library L-D-T-5is finally.

Embodiment 6

(1) L-G-1 is obtained according to the preparation method for L-G-1 inembodiment 1;

(2) L-2 is obtained according to the preparation method for L-2 inembodiment 1;

(3) L-G-2 is obtained according to the preparation method for L-G-2 inembodiment 1;

(4) L-G-2-1 is obtained according to the preparation method for L-G-2-1in embodiment 1; and

(5) the preparation of L-G-2-2.f

adding S-1, S-2 and S-3 as well as DMF into L-G-2-1, and reacting withstirring for 16 hours at 25° C.˜30° C.; filtrating the solution toremove the solvent and obtaining a filtrated cake; washing the filtratedcake respectively with distilled water and 0.1 mol/L TEAA buffersolution for 3 times to obtain L-G-2-2.f.

Specifically,

adding S-1 (FMOC-L-phenylalanine, manufacturer: Alfa, 15.1 mg), S-2(cyclohexylisocynide, manufacturer: Alfa, 4.8 mg) and S-3(cyclopentylaldehyde, manufacturer: Alfa, 4.1 mg) as well as DMF (100μL) into L-G-2-1 (20 mg), and reacting with stirring for 16 hours at 25°C.˜30° C.; filtrating the solution to remove the solvent and obtainingthe filtrated cake; washing the filtrated cake respectively withdistilled water and 0.1M TEAA buffer solution for 3 times to obtainL-G-2-2.f;

taking part of L-G-2-2-1.d (2.0 mg), adding 150 μL of strong aqueousammonia, and heating the solution to 55° C. for 1 hour to remove thesolid carrier. Removing the solvent by reducing the pressure afterfiltration process, washing the solid respectively with distilled waterand 0.1 mol/L TEAA buffer solution for 3 times. Adding 250 μL ethanoland 100 μL acetic acid-sodium acetate buffer solution (pH=4.7, 0.5mol/L) to the solid, precipitating at −20° C. to obtain DNA inL-G-2-2.f, the amount of substance of DNA in L-G-2-2.f is 35 nmol byusing OD ultraviolet absorption quantitation, the productivity is 70%.

MS(ESI) m/z 9241.9 (M+1)⁺.

(6) The preparation of L-G-3-1.f

referencing to the DNA encoded method disclosed in the patent named“Synthesis and Screening Method and Kit for Lead Compound” (applicationnumber: CN201210555548.3), L-G-2-2.f is performed DNA encoding insolid-phase condition (single strand DNA sequence isGGAGCTTGTGAAATCTGGCACTCG) to obtain L-G-2-2-1.f; after encoding, washingthe solid respectively with 0.1 mol/L TEAA buffer solution (4×100 μL)and distilled water (4×100 μL), finally washing with 100 μL distilledwater; taking part of the solid after washing, adding 50 μL of strongaqueous ammonia, and reacting for 1 hour at 55° C.; washing the obtainedsolid with 0.1 mol/L TEAA buffer solution and distilled water for 3times; precipitating the filtrated solution by ethanol, andfreeze-drying for agarose electrophoresis test;

taking part of L-G-2-2-1.f (10 mg), adding piperidine (40 μL), andreacting with stirring for 6 hours at 25° C.˜30° C.; filtrating thesolution to remove the solvent and obtaining a filtrated cake; washingthe filtrated cake respectively with distilled water and 0.1 mol/L TEAAbuffer solution for 3 times to obtain the solid; add R^(k′) (R^(k′) isselected from synthetic building block containing carboxylic acids,aldehydes or isocyanates), O-(7-azbenzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (6.1 mg, manufacturer:Alfa), DIEA (20 μL) as well as DMF (60 μL) into the solid; reacting withstirring for 16 hours at 25° C.˜30° C.; filtrating the solution toremove the solvent and obtaining a filtrated cake; washing the filtratedcake respectively with distilled water and 0.1 mol/L TEAA buffersolution for 3 times to obtain L-G-3-1-1.f;

referencing to the DNA encoded method disclosed in the patent named“Synthesis and Screening Method and Kit for Lead Compound” (applicationnumber: CN201210555548.3), L-G-3-1-1.f is performed DNA encoding insolid-phase condition (single strand DNA sequence isGGAGCTTGTGAAATCTGGCACTCG) to obtain L-G-3-1.f; after encoding, washingthe solid respectively with 0.1 mol/L TEAA buffer solution (4×100 μL)and distilled water (4×100 μL), finally washing with 100 μL distilledwater; taking part of the solid after washing, adding 50 μL of strongaqueous ammonia, and reacting for 1 hour at 55° C.; washing the obtainedsolid with 0.1 mol/L TEAA buffer solution and distilled water for 3times; precipitating the filtrated solution by ethanol, freeze-dryingfor agarose electrophoresis test; and

(7) synthesis of L-D-T-6 compound library

taking part of L-G-3-1.f (2.0 mg), adding 40 μL of aqueous ammonia, andheating the solution to 55° C. for 1 hour to remove the solid carrier.Washing the solid respectively with distilled water and 0.1 mol/L TEAAbuffer solution for several times to obtain a filtrated solution;concentrating the filtrated solution, and adding appropriate amount ofethanol, precipitating at −20° C. and centrifugating to obtain a solid;washing the solid with 85% ethanol to obtain DNA encode compound libraryL-D-T-6.

The present invention can realize synthesis of DNA coding compound inorganic solvent system. It can make the unrealized or relatively lowsynthesis of chemical reaction in the DNA coding compound library in thetraditional liquid phase condition successfully reacted, and furtherenlarge reaction types of coding compound library and the space ofdiversity of the encoded compound library. The enlarged reaction typesinclude organic catalyzed aldol reactions, alkenes metathesis reactionsetc. In the traditional liquid phase DNA encoded compound library theefficiencies are rather low, while the present invention significantlyincrease the synthesis efficiencies of these reactions.

Compared with the existed method for liquid phase synthesis of DNAencoded compound library, the synthesis technology with CPG carrying DNAencoded compound in the present invention, performs the after-reactionpurification process through several simple filtration and washingprocedures. It simplifies the after-process of chemical synthesis and ofDNA encode reactions, and simplifies the processes of separation andpurification. It also shortens the cycle of synthesis of compoundlibrary at more than 50% and saves the cost enormously. Moreover, itsignificantly enhances the purified quality, that is, enhances thepurity of final products and enhance the DNA encoding efficiency as wellas unicity.

The method for synthesis of DNA encoded compound library in the presentinvention, is able to remove the excessive DNA, decreases theinterference which excessive DNA would make on the following screeningprocess during the DNA encoded compound library synthesis and alsosignificantly increases the targeting ability and the accuracy on thescreening of new pharmaceuticals.

The linker molecules in the present invention, connect the CPG, DNA andsmall molecules at a suitable distance. Thus the forces betweenmolecules can be formed and also be broken under moderate condition,which makes a variety of reactions happens with the co-existence of DNAand CPG.

What is claimed is:
 1. A method for solid-phase synthesis of aDNA-encoded compound library, comprising: a. reacting a solid carrierG-1 with a linker molecule L-1, separating, purifying and obtainingL-G-1;

b. reacting DNA with a linker molecule L-0, separating, purifying andobtaining L-2;

c. reacting the L-G-1 with the L-2, separating, purifying and obtainingL-G-2;

d. removing a protection group of the L-G-2 and obtaining L-G-2-1; ande. adopting method 1, method 2, method 3, method 4 or any combinationthereof: method 1, comprising: reacting the L-G-2-1 with R¹, separating,purifying and obtaining L-G-2-1-1; performing DNA encoding on theL-G-2-1-1 and obtaining L-G-3-I;

or method 2, comprising: i. reacting the L-G-2-1 with skeletonmolecules, separating, purifying and obtaining L-G-2-2; and

ii. reacting the L-G-2-2 with R¹ or reacting the R¹ with the L-G-2-2having protection group removed, separating, purifying and obtainingL-G-2-2-1; performing DNA encoding on the L-G-2-2-1 and obtainingL-G-3-1;

or method 3, comprising: i. reacting the L-G-2-1 with skeletonmolecules, separating, purifying and obtaining L-G-2-2; ii. reacting theR¹ with the L-G-2-2 or reacting the R¹ with the L-G-2-2 havingprotection group removed, separating, purifying and obtaining L-G-2-2-1;performing DNA encoding on the L-G-2-2-1 and obtaining L-G-3-1; and iii.reacting a R² with the L-G-3-1 or reacting the R² with the L-G-3-1having protection group removed, separating, purifying and obtainingL-G-3-1-1; performing DNA encoding on the L-G-3-1-1 and obtainingL-G-3-2;

or method 4, comprising: i. reacting L-G-2-1 with skeleton molecules,separating, purifying and obtaining L-G-2-2; performing DNA encoding onthe L-G-2-2 and obtaining L-G-3-1; ii. reacting the R² with the L-G-3-1or reacting the R² with the L-G-3-1 having protection group removed,separating, purifying and obtaining L-G-3-1-1; performing DNA encodingon the L-G-3-1-1 and obtaining L-G-3-2; wherein the R¹ and R² aresynthetic building blocks; and f. removing the solid carrier fromL-G-3-I, L-G-3-1 and/or L-G-3-2 and obtaining DNA-encoded compoundlibrary L-D-T.
 2. The method according to claim 1, wherein in the stepa, the described solid carrier is selected from any one or more of a PEGresin, a PEGA resin, an inorganic carrier and a PE thin plate.
 3. Themethod according to claim 1, wherein in the step a, the described solidcarrier is a solid carrier containing amino active functional group; andthe solid carrier is


4. The method according to claim 1, wherein in the step a, the describedlinker molecule L-1 is selected from a compound containing any one ormore of functional groups consisting of ester group, sulfur-ester group,ortho-nitrobenzyl group, coumarin group, aromatic ketone groups,nitrine, hydroxyl group, sulfhydryl group, thioether group, carboxylgroup, aldehyde group, amino groups, amide group, alkenyl group andalkynyl group; and the described linker molecule L-1 is


5. The method according to claim 1, wherein in the step a, the describedL-G-1 is


6. The method according to claim 1, wherein in the step b, the describedDNA is single-stranded DNA.
 7. The method according to claim 1, whereinin the step b, the described linker molecule L-0 is selected from acompound containing any one or more of functional groups consisting ofester group, sulfur-ester group, ortho-nitrobenzyl group, coumaringroup, aromatic ketone groups, nitrine, hydroxyl group, sulfhydrylgroup, thioether group, carboxyl group, aldehyde group, amino groups,amide group, alkenyl group and alkynyl group; and the described linkermolecule is


8. The method according to claim 1, wherein in the step b, the L-2 is


9. The method according to claim 1, wherein in the step c, the describedL-G-2 is


10. The method according to claim 1, wherein in the step d, thedescribed L-G-2-1 is


11. The method according to claim 1, wherein in the step e, the methodof DNA encoding comprises: connecting marker sequence i with DNA ofL-G-2-1-1, L-G-2-2-1, L-G-3-1-1 or L-G-2-2 in series and marking R^(i)specifically with the marker sequence i; wherein R^(i) is a syntheticbuilding block, i=1 or 2; connecting the marker sequence i on DNA ormarker sequence (i−1) in series when a new R^(i) is added each timeduring the synthesis; and connecting a terminal sequence on the markersequence i after the synthesis is completed.
 12. The method according toclaim 1, wherein in the step e, the described R¹ and R² are respectivelyor simultaneously selected from polyfunctional group compounds, and thepolyfunctional groups are independently selected from any two or more ofgroups consisting of amino, carboxyl, aldehyde group, alkenyl, alkynyl,halogen, azide group, hydroxyl group, sulfhydryl group and phenyl group;and the described R¹ and R² are respectively or simultaneously selectedfrom amino acid, substituted or unsubstituted carboxylic acids,substituted or unsubstituted amines, substituted or unsubstitutedolefins, substituted or unsubstituted alkenes, substituted orunsubstituted alkynes, substituted or unsubstituted aldehydes,isocyanate; and the described R¹ and R² are respectively orsimultaneously selected from isocyanate, benzyl alcohol or benzoic acid.13. The method according to claim 1, wherein in the step e, thedescribed skeleton molecules contain any one or more of groupsconsisting of hydroxyl group, amino group, carboxyl group, cyanate groupand aldehyde group.
 14. The method according to claim 1, wherein in thestep e, the described skeleton molecules are selected from any one ormore of molecules consisting of 3-(4-hydroxyphenyl)propionic acid,4-aminobenzoic acid, dl-4-hydroxyphenylglycine, FMOC-glycine,FMOC-1-phenylalanine, t-butylisocynide, cyclohexyl isocyanide, 3-methylbutyraldehyde, cyclopentyl aldehyde and


15. The method according to claim 1, wherein in the step e, thedescribed L-G-3-I is

the described L-G-2-2 is

the described L-G-3-1 is:

wherein, R^(j) is a group able to form isocyanate.
 16. The methodaccording to claim 1, wherein in the step f, the method of removing thesolid carrier comprises: i. taking L-G-3-I, L-G-3-1 and/or L-G-3-2,adding an alkaline for reaction, separating and purifying; or; ii.taking L-G-3-I, L-G-3-1 and/or L-G-3-2, adding PBS buffer solution,performing decomposition reaction under light source, separating andpurifying.
 17. The method according to claim 1, wherein in the step f,the described L-D-T is

wherein, R is a group able to form isocyanate; R_(1-a) is —CH₂ or—C₆H₅CH₂CH; R_(1-b) is n-butyl, isobutyl, tertiary butyl or cyclohexyl;R_(1-a) is n-propyl, isopropyl or cyclopentyl; R^(k) is a group producedby amino group reacting with any one of carboxylic acid, aldehyde andisocyanate.
 18. A DNA encoded compound library, wherein the library hasa general formula shown as formula I:

wherein,

is a skeleton molecule of the compound library; R^(i′) is selected fromhydrogen or synthetic building blocks; E is selected from amino group,alkenyl, alkynyl, amide, ester, thioether or azide; A is selected from ahydroxyl group or a sulfhydryl group; B is selected from

wherein R is hydrogen, C1˜C8 alkyl group, C1˜C8 alkenyl or a groupforming a cycle with atoms in B₁; B₁ is selected from substituted orunsubstituted alkynyl, amino, carboxyl, aldehyde, azide or sulfhydryl;and

is DNA.
 19. The DNA encoded compound library according to claim 18,wherein the described skeleton molecule contains any one or more ofgroups consisting of hydroxyl, amino, carboxyl, cyano group and aldehydegroup.
 20. The DNA encoded compound library according to claim 19,wherein the described skeleton molecule is any one or more of followingmolecules consisting of 3-(4-hydroxyphenyl)propionic acid,4-aminobenzoic acid, dl-4-hydroxyphenylglycine, FMOC-glycine,FMOC-1-phenylalanine, t-butylisocynide, cyclohexyl isocyanide, 3-methylbutyraldehyde, cyclopentyl aldehyde and


21. The DNA encoded compound library according to claim 18, wherein themolecule A has 10˜50 atoms.
 22. The DNA encoded compound libraryaccording to claim 18, wherein the library contains the following DNAencoded compound library:

wherein, R^(j) is a group able to form isocyanate; R_(1-a) is —CH₂ or—C₆H₅CH₂CH; R_(1-b) is n-butyl, isobutyl, tertiary butyl or cyclohexyl;R_(1-a) is R_(1-a) is n-propyl, isopropyl or cyclopentyl; R^(k) is agroup produced by amino group reacting with any one of carboxylic acid,aldehyde and isocyanate.